Efficient and economic large scale purification of biomolecules such as, e.g., therapeutic proteins including antibodies, is an increasingly important consideration for the biotechnology and pharmaceutical industries. Generally, the purification processes are quite elaborate and expensive and include many different steps. For example, typically, in the case of proteins, proteins are produced using cell culture methods, e.g., using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene encoding that protein. In general, following the expression of the target protein, its separation from one or more undesired components including, e.g., host cell proteins, media by-products and DNA, poses a formidable challenge. Such separation is especially important when the therapeutic proteins are meant for use in humans and have to be approved by the Food and Drug Administration (FDA).
In general, separation and/or purification processes that are currently being used for proteins, include at least the following steps: cell lysis to recover an intracellular protein or recovery of a protein from the media in case of a secreted protein; removal of cells and cellular debris using differential centrifugation or filtration to obtain a clarified sample containing the protein of interest; and use of a variety of chromatography media in a multi-step process to separate a protein of interest from the various impurities in the sample.
Various types of polymers, including polyelectrolytes, have been employed in one or more steps for the purification of biomolecules, especially proteins. For example, the use of polyelectrolytes in flocculation to purify proteins is well established (see, e.g., International PCT Patent Application No. WO2008/091740). This can be accomplished with a wide range of polymers, with the only required general characteristic being the polymer must have some level of interaction with a species of interest (e.g., a target molecule or an impurity). The most common methodology is the use of polymers containing ion species, such as polyelectrolytes. Generally, polyelectrolytes are added to the protein mixture and purification is achieved via selective flocculation of one or more components of the mixture. A critical drawback of this approach is that carefully controlled levels of polyelectrolytes have to be added in order to avoid residual polymer contamination (e.g., when polymer level too high) or inefficient flocculation (e.g., when polymer level too low). Because ion exchange and other charged chromatography media are commonly used in the purification of proteins, residual polyelectrolytes can potentially bind to the media used in downstream purification steps, thereby fouling and complicating the process.
Recently, technology has been developed which overcomes some of the challenges associated with the use of polymers for purification of biomolecules (see, e.g., International PCT Publication No. WO 2008/079302 A2). For example, stimulus-responsive or “Smart” polymers have been developed which can bind to both soluble (e.g., host cell proteins, DNA, cell culture additives) as well as insoluble (e.g., cells and cellular debris) components (see, e.g., US Publication Nos. 20080255027 and 20090036651). Although stimulus-responsive polymers show great promise in general, a key challenge that faces a broad use of such polymers is the existence of a simple stimulus which may be implemented at a variety of scales, ranging from laboratory scale to large production scale.